Interaction between beta-adrenergic signaling and protein kinase C increases cytoplasmic Ca2+ in alveolar type II cells

The interaction between beta-adrenergic signaling and the activation of protein kinase C in alveolar type II cell plays an important role in the regulation of surfactant secretion because the combined application of beta-adrenergic agonist with protein kinase C activator to the cells stimulates the secretion synergistically. However, the mechanisms underlying the interaction are not clear. In the present study, we examined the combined effect of terbutaline with phorbol 12-myristate 13-acetate (PMA) on cytoplasmic free Ca2+ concentration ([Ca2+]i) in rat alveolar type II cells. The combined application of terbutaline with PMA to the cells rapidly increased [Ca2+]i, although neither of them affected it by itself. Similar increases of [Ca2+]i were observed in other combinations, such as terbutaline with 1-oleoyl-2-acetyl-sn-glycerol, and forskolin with PMA. Either the removal of extracellular Ca2+ or the addition of Co2+ remarkably suppressed the increase of [Ca2+]i induced by the combination of terbutaline with PMA. In addition, Co2+ inhibited the phosphatidylcholine secretion induced by the combination of terbutaline and PMA. These results suggested that the [Ca2+]i increased as a result of the interaction between formation of cyclic AMP and activation of protein kinase C in alveolar type II cells, and that the increase in [Ca2+]i was mediated by the Ca2+ influx through the plasma membrane. This mechanism to modulate [Ca2+]i may play a role in the regulation of surfactant secretion by alveolar type II cells.     

Pulmonary surfactant secretion in briefly cultured mouse type II cells

There is little information on the regulation of surfactant secretion in mouse type II cells. We isolated type II cells from C57BL/6 and FVB mice, cultured them overnight, and then examined their response to known surfactant secretagogues. Secretion of phosphatidylcholine, surfactant protein (SP)-B and SP-C was stimulated by terbutaline, 5'-N-ethylcarboxyamidoadenosine (NECA), ATP, UTP, TPA, and ionomycin. Phosphatidylcholine secretion was increased approximately twofold by all agonists in both strains of mice. The response to terbutaline and NECA is the same as in rat type II cells, whereas the response to ATP, UTP, TPA, and ionomycin is considerably less. Secretion of SP-B and SP-C was increased sevenfold by terbutaline and threefold by ATP, effects similar to those in rat type II cells. The response to terbutaline was significantly decreased in type II cells from beta(2)-adrenergic receptor null mice. These data establish that briefly cultured type II cells provide a suitable model for investigation of surfactant secretion in normal and genetically altered mice.     

Involvement of calcium in the stimulation of phosphatidylcholine secretion in primary cultures of rat type II pneumocytes by Escherichia coli lipopolysaccharide

The purpose of this study was to evaluate the mechanism by which Escherichia coli lipopolysaccharide stimulates the secretion of phosphatidylcholine in primary cultures of rat type II pneumocytes. The stimulatory effect of lipopolysaccharide on phosphatidylcholine secretion was additive to those of terbutaline and TPA (protein kinase A and C activators respectively) and this effect was not suppressed by inhibitors of both protein kinases. On the other hand, lipopolysaccharide did not modify the increase on phosphatidylcholine secretion induced by the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin, and enhanced slightly the calcium-ionophore A23187 stimulated phosphatidylcholine secretion. In addition, the stimulatory effect of lipopolysaccharide was suppressed by BAPTA, an intracellular Ca²⁺ chelator, and KN-62, a specific inhibitor of Ca²⁺-calmodulin-dependent protein kinase. These results, together with the lipopolysaccharide-mediated increase in the cytosolic [Ca²⁺], suggest that stimulation of phosphatidylcholine secretion by lipopolysaccharide in type II pneumocytes occurs by a calcium-dependent transduction mechanism via Ca²⁺-calmodulin-dependent protein kinase activation.     

Positive correlation between cytosolic free calcium and surfactant secretion in cultured rat alveolar type II cells

To determine whether increases in the cytosolic free Ca2+ concentration ([Ca2+]i) accompany agonist-stimulated surfactant secretion by cultured alveolar type II cells, we measured the [Ca2+]i of quin2-loaded cells isolated from adult rats before and after cells were stimulated with ionomycin, terbutaline or tetradecanoylphorbol acetate (TPA). To determine whether increases in [Ca2+]i are necessary for stimulated surfactant secretion to occur, we measured secretion in cells after [Ca2+]i had been reduced by loading cells with quin2 in medium containing low [Ca2+]. Ionomycin increased [Ca2+]i and stimulated surfactant secretion in a dose-dependent manner. Reductions in [Ca2+]i correlated with reductions in secretion stimulated by ionomycin, terbutaline or TPA. Ionomycin-stimulated secretion was most sensitive to reductions in [Ca2+]i; terbutaline-stimulated secretion was more sensitive than TPA-stimulated secretion. When [Ca2+]i was less than 65 nM, all stimulated secretion was blocked. Restoration of [Ca2+]i to greater than 100 nM restored ionomycin-stimulated secretion. We conclude that ionomycin increases [Ca2+]i and stimulates surfactant secretion in cultured alveolar type II cells, and that increased [Ca2+]i appears to be necessary for ionomycin-stimulated secretion to occur. Terbutaline-stimulated surfactant secretion seems to be more easily inhibited by a reduction in [Ca2+]i than does TPA-stimulated secretion.     

Stimulation of surfactant secretion by vasopressin in primary cultures of adult rat type II pneumocytes

The current study examined the effect of vasopressin on the secretion of phosphatidylcholine, the principal component of pulmonary surfactant, from adult rat alveolar type II pneumocytes in primary culture. Vasopressin stimulated secretion in a time- and dose-dependent manner. At a concentration of 10 nM, vasopressin stimulated release by 6-fold over the basal secretory rate. The concentration producing half the maximal response for vasopressin-induced secretion was 0.4 nM. The stimulation of phosphatidylcholine release by vasopressin was duplicated by the vasopressin fragment, amino acids 4 through 9. [Lys8]vasopressin and the selective vasopressin-2 agonist [deamino-8-D-Arg]vasopressin did not stimulate surfactant secretion effectively. The vasopressin- and fragment-induced secretion was inhibited by the vasopressin-1 receptor antagonist d(CH2)5TDAVP and the protein kinase C inhibitor, tetracaine, but not by the beta-adrenergic antagonist alprenolol. Vasopressin did not activate adenylate cyclase, which suggests that stimulation by vasopressin was independent of cyclic AMP. When vasopressin and isoproterenol were added concomitantly, the effects on phosphatidylcholine secretion were additive. This suggests that these two secretagogues operate via separate mechanisms.     

Developmental regulation of phospholipid secretion by fetal type II pneumocytes

Surfactant sufficiency is dependent upon adequate synthesis and secretion of surfactant by the type II alveolar epithelium. Our laboratory has previously shown that basal secretion of surfactant phospholipid by differentiated fetal type II cells is lower than the basal secretion by adult cells. The purposes of this study were to determine if undifferentiated fetal type II cells can secrete phosphatidylcholine, to determine if terbutaline, a β-adrenergic agonist, stimulates secretion of surfactant phospholipids by undifferentiated fetal cells and to examine the effects of differentiation on secretion of surfactant phospholipids by fetal cells. Constitutive (basal) secretion of phosphatidylcholine increased linearly as a function of time in both undifferentiated and differentiated cells, but the rate of secretion was greater in differentiated cells than the rate of secretion in undifferentiated cells. Terbutaline caused a concentration-dependent increase in secretion in both undifferentiated and differentiated cells. Maximal effective concentration and EC₅₀ were similar for undifferentiated (10⁻⁶ M, 0.2 μM) and differentiated (10⁻⁵ M, 0.3 μM) cells. The relative stimulation of secretion above control values was greater for undifferentiated cells. The kinetics of terbutaline stimulation varied significantly with cellular differentiation. Terbutaline resulted in 230% stimulation of secretion in undifferentiated cells at 30 min followed by a decline in the response to terbutaline at 60 to 120 min. In contrast, terbutaline stimulated secretion by differentiated cells showed a sustained linear increase from 0 to 120 min. This regulation of stimulated secretion is not present in undifferentiated cells. We conclude that undifferentiated type II cells are capable of the secretion of phosphatidylcholine and that terbutaline stimulates secretion by undifferentiated cells. Furthermore, basal secretion increases as a function of differentiation of type II cells and the regulation of stimulated secretion seen in differentiated cells is not developed in undifferentiated cells. The developmental regulation of the secretion of surfactant is complex and probably involves both excitatory as well as inhibitory mechanisms which develop at different stages of differentiation of the type II cell.     

Regulation of lung surfactant secretion by phospholipase A2

Arachidonic acid has been shown to stimulate lung surfactant secretion from alveolar epithelial type II cells. To identify the (phospho)lipases responsible for generating arachidonic acid during lung surfactant secretion, the effects of various (phospho)lipase inhibitors on phosphatidylcholine (PC) secretion from rat alveolar type II cells were investigated. N-(p-amylcinnamoyl)anthranilic acid (ACA), a general inhibitor of phsopholipase A2 (PLA2), inhibited ATP-stimulated PC secretion in a dose-dependent manner. ACA also blocked PC secretion from type II cells stimulated by other secretagogues including phorbol 12-myristate 13-acetate, Ca2+ ionophore A23187 and terbutaline, indicating that PLA2 acts at a late step distal to the generation of second messengers. To determine which PLA2 isoform(s) is involved in lung surfactant secretion, selective inhibitors to different types of PLA2 were used to inhibit PLA2 activity in type II cells. The cytosolic PLA2 (cPLA2) inhibitor, arachidonyl trifluoromethyl ketone, was found to inhibit ATP-stimulated PC secretion, whereas the secretory PLA2 inhibitors, oleoyloxyethylphosphocholine, aristolochic acid, or p-bromophenacyl bromide, and the Ca2+-independent PLA2 inhibitors, palmitoyl trifluoromethyl ketone, or haloenol lactone suicide substrate, had no effect. In addition to PLA2, arachidonic acid is released from diacylglycerol (DAG) by DAG and monoacylglycerol lipases. The DAG lipase inhibitor, RHC-80267 also blocked ATP-stimulated PC secretion. The results suggest that both pathways for generating arachidonic acid via cPLA2 and DAG lipase may participate in lung surfactant secretion.     

Suppression of cyclic AMP-dependent protein kinase activity in murine melanoma cells by 12-0-tetradecanoylphorbol-13-acetate

The effect of the potent tumor promoter 12-0-tetradeconylphorbol-13-acetate (TPA) on the cyclic AMP metabolism of B16 mouse melanoma cells was examined. TPA (10^?7M) slightly increased the growth rate and inhibited melanin production by these cells. Although TPA had little effect on basal or hormone stimulated cyclic AMP levels, it did significantly suppress cyclic AMP-dependent protein kinase activity from treated cells in a dose-dependent fashion. Other phorbol ester and non-phorbol ester tumor promoters also suppressed cyclic AMP-dependent protein kinase activity while the non-promoter, phorbol, did not alter cyclic AMP-dependent protein kinase activity.     

Surfactant protein A regulates pulmonary surfactant secretion via activation of phosphatidylinositol 3-kinase in type II alveolar cells

Pulmonary surfactant is secreted by the type II alveolar cells of the lung, and this secretion is induced by secretagogues of several types (e.g., ionomycin, phorbol esters, and terbutaline). Secretagogue-induced secretion is inhibited by surfactant-associated protein A (SP-A), which binds to a specific receptor (SPAR) on the surface of type II cells. The mechanism of SP-A-activated SPAR signaling is completely unknown. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 rescued surfactant secretion from inhibition by SP-A. In order to directly demonstrate a role for PI3K in SPAR signaling, PI3K activity was immunoprecipitated from type II cell extracts. PI3K activity increased rapidly after SP-A addition to type II cells. Since many receptors that activate PI3K do so through tyrosine-specific protein phosphorylation, antisera to phosphotyrosine, insulin-receptor substrate-1 (IRS-1), or SPAR were also examined. These antisera coimmunoprecipitated PI3K activity that was stimulated by SP-A. In addition, the tyrosine-specific protein kinase inhibitors genistein and herbimycin A blocked the action of SP-A on surfactant secretion. We conclude that SP-A signals to regulate surfactant secretion through SPAR, via pathways that involve tyrosine phosphorylation, include IRS-1, and entail activation of PI3K. This activation leads to inhibition of secretagogue-induced secretion of pulmonary surfactant.     

Interactions between cyclic AMP- and phorbol ester-dependent phosphorylation systems in S49 mouse lymphoma cells

High-resolution two-dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP-dependent protein kinase or the guanyl nucleotide-binding "Ns" factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild-type and mutant cells: Phosphorylations shown previously to be responsive to activation of intracellular cyclic AMP-dependent protein kinase were stimulated by TPA in wild-type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild-type cells, and two novel phosphoprotein species appeared in response to TPA only in wild-type cells. The latter two classes of TPA-mediated responses specific to wild-type cells could be evoked in adenylate cyclase-deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: 1) TPA stimulates adenylate cyclase in wild-type cells causing increased phosphorylation of endogenous substrates by cyclic AMP-dependent protein kinase, 2) activated cyclic AMP-dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA-dependent phosphoproteins, and 3) cyclic AMP-dependent events facilitate TPA-dependent phosphorylation of some substrate proteins.     

Regulation of surfactant secretion from isolated Type II pneumocytes by substance P

Substance P, an eleven amino acid neuropeptide, significantly inhibited release of [3H]phosphatidylcholine from pulmonary Type II epithelial cells in vitro. Basal release and release in response to the beta-adrenergic agonist, terbutaline and 12-O-tetradecanoylphorbol 13-acetate (TPA) were significantly decreased in the presence of substance P. Inhibitory effects of substance P were noted following a 1 h exposure of primary cultures of Type II cells in vitro and persisted up to 3 h in the presence of the secretagogues, TPA and terbutaline. The IC50 values for substance P inhibition of [3H]PC release were 10 microM for basal release, 40 microM for TPA-induced release and 50 microM for terbutaline-induced release. The related neuropeptide, physalaemin and the stable active analog of substance P, [pGlu5, MePhe8, MeGly9]substance P [5-11], had no significant inhibitory effects on surfactant release whether in the presence or absence of TPA or terbutaline. These data support the hypothesis that NH2-terminal basic groups of substance P are necessary for inhibition of surfactant secretion from isolated Type II cells and support the concept that an inhibitory system contributes to mediation of surfactant secretion from Type II epithelial cells.     

Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C

Bombesin caused a marked stimulation of 32Pi into phosphatidylinositol (PI), with no apparent lag, and into phosphatidylcholine (PC), after a lag of about 20 min. Stimulation was blocked by the bombesin receptor antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P, indicating that the effects on both PI and PC were mediated through the same receptor. The tumor-promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and dioctanoylglycerol (diC8) both directly activate protein kinase C and in this report were shown to stimulate 32Pi incorporation into PC but not into Pl. In addition, TPA stimulated the release of [3H]choline and [3H]phosphocholine and the accumulation of [3H]diacyglycerol from prelabelled cells. These results strongly suggest that TPA activates a phospholipase C specific for PC. Pretreatment of cells with phorbol-12, 13-dibutyrate (PDBu) for 24 h depleted cellular protein kinase C activity and inhibited the ability of TPA to induce these effects suggesting a direct involvement of protein kinase C. Similarly the bombesin stimulation of 32Pi into PC and of [3H]choline and [3H]phosphocholine release was inhibited by PDBu pretreatment. DiC8 and, to a lesser extent, TPA stimulated the translocation of CTP:phosphocholine cytidylytransferase from the cytosolic to the particulate fraction. DiC8 also stimulated this translocation in cells depleted of protein kinase C. It was concluded that both bombesin and TPA activated protein kinase C leading to activation of a phospholipase C specific for PC.     

Thyrotropin-releasing hormone and phorbol esters induce phosphatidylcholine synthesis in GH3 pituitary cells. Evidence for stimulation via protein kinase C

Phorbol esters have been shown to stimulate phosphatidylcholine synthesis via the CDP-choline pathway. The present study compares the effects of phorbol esters and thyrotropin-releasing hormone (TRH) on phosphatidylcholine metabolism in GH3 pituitary cells. In a previous study (Kolesnick, R.N., and Paley, A.E. (1987) J. Biol. Chem. 262, 9204-9210), the potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced time- and concentration-dependent incorporation of 32Pi and [3H]choline into phosphatidylcholine in short-term labeling experiments. In this study, TPA is shown to activate choline-phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme of the CDP-choline pathway, by stimulating redistribution of the inactive cytosolic form of the enzyme to the membrane. Redistribution was quantitative. TPA reduced cytosolic activity from 3.5 +/- 0.4 to 1.5 +/- 0.3 nmol . min-1 x 10(7) cells-1 and enhanced particulate activity from 2.5 +/- 0.4 to 4.9 +/- 0.6 nmol . min-1 x 10(7) cells-1. TRH also stimulated time- and concentration-dependent 32Pi and [3H]choline incorporation into phosphatidylcholine. An increase was detectable after 5 min; and after 30 min, the levels were 164 +/- 9 and 150 +/- 11% of control, respectively; EC50 congruent to 2 X 10(-10) M TRH. These events correlated directly with TRH-induced 32Pi incorporation into phosphatidylcholine. TRH also stimulated redistribution of cytidylyl-transferase specific activity. TRH reduced cytosolic activity 45% and enhanced particulate activity 51%. Neither TRH nor TPA stimulated phosphatidylcholine degradation. In cells down-modulated for protein kinase C (Ca2+/phospholipid-dependent protein kinase), the effects of TPA and TRH on 32Pi incorporation into phosphatidylcholine were abolished. However, TRH-induced incorporation into phosphatidylinositol still occurred. These studies provide evidence that hormones may regulate phosphatidylcholine metabolism via the protein kinase C pathway.     

Functional evidence for involvement of P2 purinoceptors in the ATP stimulation of phosphatidylcholine secretion in type II alveolar epithelial cells

There is evidence that phosphatidylcholine secretion in type II pneumocytes is stimulated by adenosine and adenine nucleotides and that the effect of adenosine is mediated by the A2 subtype of the P1 purinoceptor. To determine if the effect of ATP is also mediated by the same receptor following its catabolism to adenosine or by the P2 purinoceptor we compared the effects of adenosine and ATP. Adenosine and terbutaline stimulated phosphatidylcholine secretion approx. 2-fold, while ATP stimulated it by more than 3-fold, essentially to the same extent as the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate. The stimulatory effect of adenosine but not of ATP was abolished by adenosine deaminase. The effect of ATP was markedly diminished by the P2 desensitizing agent alpha,beta-methylene ATP, but only slightly by the P1 antagonist 8-phenyltheophylline. Adenosine increased the cAMP content of type II cells while ATP had little effect. The effects of ATP and terbutaline were additive while those of adenosine and terbutaline were not. These data show that ATP and adenosine stimulate phosphatidylcholine secretion via different mechanisms. Therefore, the effect of ATP is not mediated via catabolism to adenosine. Metabolically resistant analogs of ATP also stimulated secretion in a concentration-dependent manner although none were as potent as ATP. The order of potency was ATP greater than beta,gamma-methylene ATP = 2-methylthio ATP = 2-deoxy ATP greater than or equal to 8-bromo ATP greater than alpha,beta-methylene ATP. The facts that ATP analogs also stimulate secretion and that the effect of ATP was antagonized by alpha,beta-methylene ATP suggest that the stimulatory effect of ATP is mediated by the P2 purinoceptor.     

Gonadotropin release and redistribution of calcium-activated, phospholipid-dependent protein kinase in phorbol-stimulated rat pituitary cells

The effect of phorbol esters on calcium-activated, phospholipid-dependent kinase (protein kinase C) and luteinizing hormone (LH) secretion was examined in cultured rat anterior pituitary cells. The potent tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) stimulated LH secretion and activated pituitary protein kinase C in the presence of calcium and phosphatidylserine. The enzyme activity present in cytosol and particulate fractions was eluted at about 0.05 M NaCl during DE52-cellulose chromatography. Preincubation of pituitary cells with TPA markedly decreased cytosolic protein kinase C activity and increased enzyme activity in the particulate fraction. The maximal TPA-induced change in enzyme activity, with a 76% decrease in cytosol and a 4.3-fold increase in the particulate fraction, occurred within 10 min. The dose-dependent changes in protein kinase C redistribution in TPA-treated cells were correlated with the stimulation of LH release by the phorbol ester. These results suggest that activation of protein kinase C by TPA is associated with intracellular redistribution of the enzyme and is related to the process of secretory granule release from gonadotrophs.     

Tetradecanoylphorbol acetate and terbutaline stimulate surfactant secretion in alveolar type II cells without changing the membrane potential

Alveolar type II cells were isolated from adult rat lungs after tissue dissociation with elastase. The effect of known secretagogues on transmembrane potential was examined in freshly isolated cells (day 0 cells) and in cells after one day of primary culture (day 1 cells). Freshly isolated type II cells were incubated with 3,3'-dipentyloxacarbocyanine (di-O-C5(3)) or 3,3'-dipropylthiadicarbocyanine (di-S-C3(5)), dyes whose intracellular fluorescence intensity is a direct function of the cellular transmembrane potential. Fluorescence was continuously recorded by fluorescence spectrophotometry. Type II cells rapidly incorporated the dyes, and the addition of gramicidin (1 microgram/ml) depolarized the cells as indicated by a change in fluorescence. Neither 12-O-tetradecanoylphorbol 13-acetate (TPA) nor terbutaline plus 3-isobutyl-1-methylxanthine (IBMX), which stimulate surfactant secretion from isolated alveolar type II cells, changed the transmembrane potential. The lipophilic cation triphenylmethylphosphonium (TPMP+) was used to quantitate the transmembrane potential of type II cells cultured for one day. Addition of TPA or terbutaline plus IBMX induced surfactant secretion but did not alter the transmembrane potential. To study further the relationship of secretion to the transmembrane potential, secretion was also determined in the presence of high extracellular potassium which depolarizes the cells and in the presence of choline in place of sodium. High potassium enhanced the basal secretion of phosphatidylcholine from 1.8% to 3.4% (P less than 0.01, n = 7). Substitution of sodium chloride by choline chloride had no effect on basal secretion but enhanced TPA-induced secretion (P less than 0.01). We conclude that high extracellular potassium induces membrane depolarization and stimulates surfactant secretion, but TPA or terbutaline plus IBMX stimulates secretion without detectable membrane depolarization and stimulation of secretion by TPA does not require extracellular sodium.     

The Na+/H+ antiporter in rat alveolar type II cells and its role in stimulated surfactant secretion

We used the pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to identify Na+/H+ exchange in freshly isolated rat alveolar type II cells and alveolar type II cells in primary culture. The intracellular pH (pHi) of freshly isolated alveolar type II cells was 7.36 +/- 0.05 (n = 3). When freshly isolated alveolar type II cells were acid loaded with nigericin in sodium-free buffer, the pHi dropped to 6.59 +/- 0.04 and remained low in sodium-free buffer. When acid-loaded cells were subsequently incubated with NaCl, pHi increased in a dose-dependent manner. Amiloride (0.1 mM) inhibited the sodium-induced increase in pHi. When the acid-loaded cells were resuspended in an unbuffered choline chloride solution, the cells secreted H+ in a sodium-dependent and amiloride-inhibitable manner. Alveolar type II cell monolayers, which were cultured for 22 h on glass coverslips and then loaded with BCECF, had a resting pHi of 7.48 +/- 0.05 (n = 4). Nigericin acidified these cultured cells in the absence of sodium and NaCl increased the pHi of these acid loaded cells as observed in freshly isolated cells. Secretagogues of pulmonary surfactant, 12-O-tetradecanoylphorbol 13-acetate (TPA) and terbutaline, did not change pHi. Inhibition of the Na+/H+ antiporter by the addition of amiloride to a Na+ containing medium or the substitution of choline for Na+ did not inhibit stimulated phosphatidylcholine secretion. We conclude that pHi regulation in rat alveolar type II cells is in part mediated by an amiloride-sensitive Na+/H+ antiporter, but this system appears not to be involved in TPA- or terbutaline-induced pulmonary surfactant secretion in primary culture.     

Pulmonary surfactant secretion is regulated by the physical state of extracellular phosphatidylcholine.

Pulmonary alveolar type II cells synthesize, secrete, and recycle the components of pulmonary surfactant. In this report we present evidence that dipalmitoylphosphatidylcholine is a potent inhibitor of surfactant lipid secretion by type II cells. Monoenoic and dienoic phosphatidylcholines with fatty acids of 16 or 18 carbons are ineffective as inhibitors of surfactant lipid secretion. In contrast, disaturated phosphatidylcholines, with either symmetric or asymmetric pairs of fatty acids of 14, 16, or 18 carbons, exhibit inhibition of surfactant secretion that correlates extremely well with the phase transition temperature (Tc) of the phospholipid. The inhibitory activity of dipalmitoylphosphatidylcholine is not dependent upon lipid stereochemistry. N-Methylated derivatives of dipalmitoylphosphatidylethanolamine are significantly less effective than phosphatidylcholine as inhibitors. Phosphatidylcholines below their phase transition temperature are inhibitors of surfactant secretion, whereas those above their phase transition temperature are either ineffective or weakly inhibitory. The phase transition dependence of inhibition is observed when type II cells are incubated at 37 degrees C with different species of phosphatidylcholine. In addition, if type II cells are stimulated to secrete at different temperatures the efficacy of a given phospholipid as an inhibitor is dependent on its relationship to Tc (i.e. dipalmitoylphosphatidylcholine with a Tc of 41 degrees C significantly inhibits secretion at 37 degrees C but not at 42 degrees C). Inhibition of surfactant secretion by dipalmitoylphosphatidylcholine is abrogated when it is incorporated into the same liposome with dioleoylphosphatidylcholine as a 50:50 mixture. In contrast, the simultaneous addition of two separate populations of liposomes, one composed of dipalmitoylphosphatidylcholine and the other composed of dioleoylphosphatidylcholine, does not significantly alter the inhibitory activity found with dipalmitoylphosphatidylcholine alone. These data provide compelling evidence that the physical state of phosphatidylcholine can regulate surfactant secretion from alveolar type II cells and suggest a unique mechanism for regulating exocytosis in the alveolus of the lung.     

Differential activation of type-I and type-II adenosine 3'':5''-cyclic monophosphate-dependent protein kinases in liver of glucagon-treated rats.

The protein-bound cyclic AMP and the activity of cytosolic protein kinases in the presence and absence of cyclic AMP were determined in rat liver up to 2h after injection of glucagon. On the basis of the different salt-sensitivities of the activated cyclic AMP-dependent proteinkinases I and II, an activation of protein kinase II restricted to the high cyclic AMP concentrations present in the first 30 min after hormone injection was found. Essentially the same result was obtained by chromatographic analysis on DEAE-cellulose of liver cytosol from untreated rats and from rats killed at 2 and 60 min after glucagon injection. Protein kinase II activation was only detected at 2 min after injection. In contrast, the cyclic AMP-dependent protein kinase I was found to be nearly totally activated at 2 min and to be still almost as active at 60 min after the hormone stimulus, whereas the amount of bound cyclic AMP and the activation of total cytosolic protein kinases had fallen to two-thirds of their maximal values during this time period. A third cyclic AMP-independent protein kinase, which co-chromatographed with protein kinase type II, could be clearly distinguished from the two cyclic AMP-dependent kinases by use of the heat-stable inhibitor from bovine muscle, which totally inhibited the cyclic AMP-dependent enzymes, but stimulated the cyclic AMP-independent protein kinase.     

Dissociation of protein kinase C activation from phorbol ester-induced maturation of HL-60 leukemia cells

The role of C-kinase in the induction of maturation of HL-60 promyelocytic leukemia cells was examined using two activators of this kinase, 12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG). At 10(-8) M, a concentration that induced maturation, TPA effectively stimulated C-kinase activity in cell-free preparations by increasing the affinity of the enzyme for Ca2+. Similar activation was observed with 20 micrograms/ml of OAG. At these concentrations, addition of either compound to intact cells stimulated the phosphorylation of cellular proteins. Treatment with TPA resulted in an increased phosphorylation of 14 proteins, 9 of which also changed in response to OAG. In addition to the effects on protein phosphorylation, TPA and OAG both affected choline lipid metabolism. TPA at 10(-8) M stimulated the incorporation of [methyl-3H]choline into phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine. OAG at 20 micrograms/ml had quantitatively similar effects on the labeling of the former two lipids, but did not affect incorporation of choline into lysophosphatidylcholine. Despite the similar biochemical effects of TPA and OAG, the diglyceride was unable to induce HL-60 cell maturation as measured by inhibition of cell growth, development of nonspecific esterase activity, phagocytosis, adherence of cells to plastic, and loss of transferrin receptor activity. The lack of effect is not due to metabolism of OAG; maturation could not be induced by treating cells with fresh OAG every 2 h for a period of 12 h. These results suggest a dissociation of the activation of C-kinase and the induction of HL-60 cell maturation by TPA.